Some of the processes by which a cell utilizes its genetic material to direct its growth, differentiation, and other functions include transcription, translation, and replication. Transcription is the process by which DNA is converted into RNA for subsequent translation into a protein. Replication is the process by which DNA sequences are duplicated prior to cell division. These processes represent the principle activities localized in the cell nucleus.
Transcription is a complex process that controls gene expression and largely determines the actions and properties of cells. Most eukaryotic genes are regulated by multiple transcription-control elements, specific DNA sequences lying outside of the coding region of the gene. Transcription-control elements serve as specific binding sites for a variety of protein transcription factors, both activators and repressors.
In general, transcription activators are believed to control the assembly of the transcription initiation complex and the rate at which the poised RNA polymerase in this complex initiates transcription. The common feature among transcription activators is that each binds to one or several specific DNA regulatory elements, then participates in moderating the activity of the transcriptional apparatus. Generally transcriptional activators act in response to a cascade of intracellular reactions initiated by an extracellular signal. Although capable of acting alone, transcription activators are also known to interact with additional protein components, called coactivators, that can modify their activities. Well known transcription activators include the nuclear receptors, NF-.kappa.B, cAMP-response element binding protein (CREB), and the STATs.
The nuclear receptors, also known as steroid/retinoid hormone receptors, are transcription factors that exist in an inactive form either in the cytoplasm or nucleus. Upon binding their respective hormonal ligands, the receptors undergo activation or "transformation," and the activated receptor can then effectively bind to DNA and activate transcription of a cis-linked gene. Well known examples of nuclear receptors include the glucocorticoid receptor (GR), the retinoid receptors (RAR and RXR), the estrogen receptor (ER), the mineralocorticoid receptor (MR), the androgen receptor (AR), the progesterone receptor (PR), peroxisome proliferator-activated receptors (PPAR), vitamin D receptor (VDR), thyroid hormone receptor (THR) and the like. Reviews discussing the structure and function of nuclear receptors are found in Wahli and Martinez, FASEB J 5:2243-2249, 1991; Evans, Science 240:889-895, 1988; and Tsai and O'Malley, Annu. Rev. Biochem. 63;451-486, 1994, all of which are hereby incorporated by reference in their entirety. Nuclear receptor activity effects such diverse cellular functions as differentiation, proliferation and inflammatory response.
Nuclear factor-kappa B (NF-.kappa.B) encompasses a family of inducible transcriptional activators critical in the regulation of gene expression in the lymphocytic response to injury and inflammatory stimuli. In the cell, NF-.kappa.B exists as a homo- or heterodimer with distinct DNA binding specificities. Cellular stimulation with inflammatory cytokines, such as the interleukins, and other inflammatory stimuli results in the rapid translocation of NF-.kappa.B to the nucleus where it binds to specific .kappa.B elements. Many genes involved in the inflammatory response are induced by NF-.kappa.B including pro-inflammatory cytokines, chemotactic proteins and adhesion molecules. Steroids and retinoids are widely reported to exert anti-proliferative and anti-inflammatory effects in T lymphocytes in part by repressing the activities of NF-.kappa.B (and AP-1). In turn, NF-.kappa.B activity represses nuclear receptor function.
Elevation in the level of cytosolic cAMP, such as in response to G-coupled protein receptor activity, induces the expression of many genes. These genes are under the control of a cAMP-response element (CRE), essential for their induction, which is bound by the transcription activator CREB (cAMP-response element binding protein). CREB activity is promoted by its phosphorylation, which recruits the coactivator CREB-binding protein (CBP/p300). CBP can act as a coactivator for a number of other transcription activators including c-Jun, c-Myb, c-Fos, NF-.kappa.B and nuclear receptors, among others. CBP/p300 interacts with the transcription factor and tumor suppressor p53 and modulates its activity (Avantaggiati, et al Cell 89:1175-1184, 1997). In addition to its activity as a transcription factor, CBP enzymatically acetylates histones and other proteins, such as p53. This acetylation activity promotes CBP-dependent regulation of chromatin functioning, (Ogryzko, et al. Cell 87:953-959, 1996). Thus, CBP may perform an important role in the integration of diverse signaling pathways that result in changes in gene expression.
The cytokine-receptor superfamily includes receptors for protein ligands such as the interferons, erthryropoetin, and the like. Transcriptional activation in response to these receptors involves the phosphorylation and subsequent dimerization of a family of transcriptional activators called the STATs (Signal Transducers and Activators of Transcription). STATs can form homo- or heterodimers and bind to a number of DNA sequences including the interferon-stimulated response-element (ISRE) and the serum-inducible response element (SRE). STAT activity can also be induced by the activity of some tyrosine kinase receptors, such as the epidermal growth factor receptor. STAT activity regulates the expression of therapeutically important genes. In response to the binding of interferon, for example, cells are induced to express a set of proteins that make them resistant to viral infection.
Recent research has uncovered new evidence for the localization of several nuclear activities in substructures that may contribute a topological function to these processes. The nuclei of cells contain a variety of substructures among which are several morphologically distinct types, called nuclear bodies, including sphere organelles and coiled bodies. A third type of nuclear body having a distinct nuclear topology has recently been described, and is variously referred to as the PML Oncogenic Domain (POD), Nuclear Domain (ND) 10, or Kr body. PODs and POD-localized proteins have been proposed as playing a role in transcription and may be involved in translation, replication and nuclear transport as well. PODs have been found in all the cell types examined to date, suggesting that they are a fundamental structure common to all cells.
PODs are comprised of a number of cellular proteins, all of unknown function, arranged in a vesicle-like structure around an electron dense core. Among these proteins are PML, Sp-100, PIF13, PIF31, ND52, ND55, and Isg20 (Doucas and Evans, Biochimica et Biophysica Acta 1288:M25-M29, 1996; Gongora, et al, J Biol Chem 272(31):19457-19463, 1997). Several of these proteins (e.g., PML, Sp-100) are the targets of antibodies present in the serum of patients with autoimmune disorders such as primary bilary cirosis and systemic lupus erythramatosis (Andre, et al, Exp Cell Res 229:253-260, 1996).
A chief component of PODs is the protein PML. PML was originally identified by its role in Acute Promyelocytic Leukemia (APL), a disorder characterized by a chromosomal translocation (15;17) which fuses PML to the retinoic acid receptor resulting in a PML:RAR fusion product that retains most of the functional domains of its parental proteins.
PML contains several different protein:protein interaction domains including a cysteine-rich domain containing a RING finger motif and a coiled-coil or alpha-helical region. The presence of protein:protein interaction domains predicts that PML possesses a self-polymerizing feature that permits efficient packaging and transfer of bound target molecules into a round vesicle-like structure. In fact, PML:RAR forms homodimers and PML:RAR-PML heterodimers in vitro, which results in disruption of normal POD structure and may contribute to the oncogenic state in APL patients. Treatment with retenoic acid results both in restoration of POD structure and cellular differentiation with subsequent clinical remission.
POD structure has also been linked with viral replication. Doucas, et al have shown that infection with adenovirus disrupts POD structure (Doucas, et al, Genes & Dev. 10:196-207, 1996), as is also the case when cells are genetically engineered to express certain adenovirus proteins. POD disruption can be reduced, however, by an increase in the expression of PML, which also reduces viral replication. These results suggest that adenovirus replication may utilize POD components. It has not been demonstrated, however, that POD-associated proteins directly contribute to transcriptional/translational/replication control nor has a role for PODs been shown using clinically relevant viruses.
A variety of serious pathogenic disorders involve transcription and translation at some level. For example, the process of inflammation requires the transcription and translation of a variety of proteins such as inflammatory cytokines and chemokines, and the like. Current anti-inflammatory treatments strive to ameliorate the undesirable effects of these proteins with varying degrees of success, often producing severe side effects. A substance capable of regulating the transcription and/or translation of inflammatory proteins could modulate the inflammatory process without the side effects of current treatments.
Cancer is another pathogenic disorder that involves transcriptional and translational regulation. Disruption of normal transcription can result in an inability of cells to differentiate, for example, a hallmark of the cancerous state. Restoration of transcription would allow these cancer cells to resume a normal differentiation pathway, ultimately ending in apoptosis. To date, however, many, if not most treatments for cancer involve the use of cytotoxic agents which target and kill any rapidly dividing cell, including those that are perfectly normal. Therefore, a need still exists for new methods of treating cancer that are more selective and have fewer side effects.
Viral infections, such as with the human immunodeficiency virus, can have a severe debilitating effect on the infected host. Viruses themselves are simple organisms lacking the full range of transcriptional machinery necessary for their efficient propagation in the cell. Most viruses rely on their ability to co-opt the transcriptional/translational apparatus of their hosts in order to multiply. The anti-viral therapies currently in use often target viral specific proteins, such as viral proteases. The rapid replication and mutation rate of most viruses, however, often allows new strains to emerge that are resistant to these therapies.
Thus a need exists for new substances that can be used to treat these and other pathogenic disorders. The invention disclosed herein addresses that need.